1. Field of the Invention
The present invention deals with novel chemical conjugates for the selective delivery of cytotoxic drugs to tumor cells, and with methods of destroying tumor cells using such conjugates.
2. Background of the Invention
It is well known that current cancer therapy involves the use of antimitotic drugs such as adriamycin, vincristine, cisplatin, daunomycin and methotrexate, which all have strong undesirable side-effects on the normal cells of the patient. It is therefore important that the activity of antitumor drugs be specifically directed to the malignant cells and have little toxic effect on the normal cells.
One approach to selectively delivering cytotoxic agents to tumor cells requires use of antibodies which preferentially bind tumor-associated or tumor-specific antigens such as alpha fetoprotein. The antibody may be radiolabeled, or it may be conjugated to toxins such as hematoporphyrin, abrin, ricin, diphtheria toxin, Pseudomonas exotoxin, gelonin or to the above-mentioned antimitotic drugs. For reviews see K. Sikora et al., (1984), Br. Med. Bull., 40, 233-9; and P. C. Thorpe, et al., (1985), in Monoclonal Antibodies B4, Biological & Clinical Applications. Further, Pastan, U.S. Pat. No. 4,545,985 discloses the binding of Pseudomonas exotoxin to antibodies, for instance antibodies to specific human cell receptors such as the transferrin receptor.
There are a number of disadvantages in the use of antibodies as targeting agents, as described by I. Pastan, et al., (1986), Cell, 47, 641-648. First, the internalization of antibody conjugates into cells is highly variable, depending on the antibody and the cell. Second, the antibody may itself be antigenic and stimulate an immune response in the patient, which would limit the effectiveness of the conjugate. Third, the antibody may bind to normal cells, which do not have the antigen recognized by the antibody, by means of Fc receptors that occur on many nontumor cells and can react with many antibodies. Fourth, it is difficult to attach a large number of drug molecules to an antibody without adversely affecting its antigen-binding activity [Hurwitz, et al., (1975), Cancer Res., 35, 1175-1181]. Finally, the high molecular weight of antibodies reduces their ability to penetrate into tumors between the cells [See Delabye, et al., (1986), J. Clin. Invest. 77: 301-11; Buchegger, et al. (1983), J. Exp. Med 158: 413-27; Buchegger et al. (1986), Cancer,58, 655-61.]
Another approach is to attach the drug to a polyaminoacid carrier which reduces its cytotoxic action in normal cells. This carrier may bear a large number of drug molecules. In the drug-carrier complex, the amino acid composition or sequence is intended to provide preferential release of the cytotoxin at tumor cells by means of degradative enzymes known to be present in elevated concentrations in tumor cells. For example, Zunino, et al., (1982), Int. J. Cancer 30, 465-70, reports on the antitumor activity of daunorubicin linked to poly-L-aspartic acid, and Kato, et al., (1984), Cancer Res. 44, 25, describes antitumor activity of cytosine arabinoside conjugated with polyglutamic acid. W.A.R. van Heeswijk, et al., (1985), J. Controlled Release 1, 301-315, and Hoes et al., (1985), J. Controlled Release 2, 205-213, describe additional conjugates of polyglutamic acid backbones with peptide spacers to which adriamycin is covalently coupled.
The combination of a tumor-directed antibody and a degradable polyaminoacid carrier with a cytotoxic drug has also been described. For example, Kato, et al., (1984), J. Medicinal Chem. 27, 1602-1607, reports conjugation of daunomycin (DM) to polyglutamic acid (PGA) and coupling the resulting cytotoxic polymer with rat alpha fetoprotein (AFP) antibody. Upon trial, the cytotoxic activity of the resulting anti-AFP-PGA-DM conjugate was shown to be more effective than nIg (a control antibody), anti-AFP, unconjugated DM, PGA-DM or nIg-PGA-DM.
Similarly, EP-A 112,720 (Teijin) discloses a conjugate comprising an immunoglobulin capable of binding selectively to a particular antigen possessed by a cell to be killed, a polymer carrier and a cytotoxic substance linked thereto, for instance p-(N,N-bis(2-Chloroethyl))-phenylenediamine, melphalan, 1-(beta-D-arabino-furanosyl) cytosine and its phosphate, methotrexate, actinomycin D, mitomycin C and the like, while Runge, U.S. Pat. No. 4,485,093 discloses an immunotoxin conjugate for treating malignant diseases, which consists of arsanilic acid and tumor specific antibody covalently bound to a polyglutamic acid back-bone.
Unfortunately, the use of a polyaminoacid carrier does not solve all of the problems associated with the use of immunotoxins. In addition to the problems related to using antibodies directly conjugated to cytotoxins, addition of such carriers to antibodies, or even smaller Fab fragments, may undesirably further reduce their ability to penetrate tumors efficiently.
Pastan, U.S. Pat. No. 4,545,985 suggested conjugating Pseudomonas exotoxin (PE) to a variety of peptides, proteins and growth factors that react with specific receptors on cells, including sarcoma growth factors, melanocyte stimulating hormone, somatostatin, glucagon, insulin, transferrin, low density lipoprotein (LDL), calcitonin, alpha-2-macroglobulin and lysine bradykinin. Pastan constructed a conjugate of Pseudomonas exotoxin (PE) and a peptide hormone isolated from mice, Epidermal Growth Factor (EGF), by introducing thiol groups into each and then linking the two using a disulfide exchange reaction. The conjugate was toxic to KB tumor cells, but nude mice injected with the conjugate died of liver failure. The coupling destroyed the toxin's ability to bind to its own receptor, so toxicity was mediated by the EGF receptors of the liver cells.
N. Shimizu, et al., (1980), FEBS Letters 118, 274-278, reported the preparation of a covalent conjugate of EGF and diphtheria toxin. In addition, Cawley and Herschman, (1980), Cell 22, 563-570, and Simpson, et al., (1982), Cell 29, 469-673, reported on conjugates of EGF covalently linked to the polypeptide A chain of ricin, or to diphtheria toxin, by a disulfide bridge. These conjugates containing EGF generally showed high but unpredictably variable toxicity for various normal cells.
[For techniques of preparing or isolating EGF from various species, including the human form (also called urogastrone), or other variants or analogues thereof, and for discussion of their properties, see W085/00369 (Chiron); W083/04030 (Applied Molecular Genetics); Nishimura, U.S. Pat. No. 4,528,186; EP-A 46,039 (G.D. Searle); EP-A 128,733 (Genentech); Komoriya, W085/01284; Wakanuga, EP-A 131,868; and Camble, U.S. Pat. No. 3,917,824.]
More recently, additional similar conjugates of non-immunoglobulin peptides and cytotoxins have been reported. For example, Bacha, et al., (1983), J. Biol. Chem. 258, 1565-1570, describes conjugates of thyrotropin-releasing hormone (TRH) and polypeptides related to diphtheria toxin, which were toxic to pituitary tumor cells; and O'Keefe and Draper, (1985), J. Biol. Chem., 260, 932-937, report on characterization of a transferrin-diphtheria toxin conjugate, which was toxic to mouse cell cultures. Furthermore, Murphy, et al., (1986), Proc. Nat. Acad. Sci.,
USA, 83, 8258-8262, describes melanoma-selective cytotoxicity of a genetically fused protein which is equivalent to a covalent conjugate of the alpha melanocyte-stimulating hormone (MSH) and a cytotoxin related to diphtheria toxin. JP 60163824 (Nippon Shinyaku KK) discloses a drug carrier, wherein the carrier comprises a protein, such as an apolipo-protein obtained from a serum lipoprotein (e.g., LDL), and a lipid, designed for selectively carrying the pharmaceutical ingredient to the necessary tissue.
Again, the number of toxin molecules that may be directly attached, as taught above, to a molecule of a peptide hormone or growth factor is limited by the need to preserve receptor binding activity. Furthermore, such conjugates of a non-immunoglobulin and a cytotoxin may be excessively toxic for normal cells bearing the appropriate receptors.
No admission is made that any of the foregoing references constitute "prior art" and all descriptions are based on the publications rather than on firsthand knowledge of the work described.